Preparedness and competency of New Zealand graduates for general dental practice - perceptions from the workforce.

BACKGROUND: Dental graduates need to demonstrate clinical competency. This mixed-methods study explored the perceptions of clinicians who employ or work with new graduates from the University of Otago, New Zealand, and identified themes reflecting graduates' preparedness for independent practice. METHODS: An online survey using a semantic differential scale and open-ended questions collected opinions and experiences from the workforce. Quantitative data were analysed using SPSS software, and qualitative data were analysed thematically. RESULTS: A representative sample of the workforce was obtained with a response rate of 35% (N = 83). Most clinicians engage new graduates to support the profession and/or rural communities. They perceived that graduates were well prepared in most areas, could translate theory to clinical practice and demonstrate professionalism. Graduates were reportedly stronger in basic dentistry, communication, ethics, and record keeping however were less strong in complex treatment planning, molar endodontics, fixed prosthodontics and exodontia. Clinical exposure during dental training was perceived as more limited, and mentoring and guidance in the transition to practice were deemed to be important. CONCLUSIONS: New Zealand dental graduates appear prepared for independent practice; however, maximising clinical opportunities during training, mentoring and early professional development in advanced areas of practice is essential to enhance competency and confidence.

Storage-related stability and antimicrobial efficacy of bottled, neutral-pH Electrolysed Oxidising Water.

OBJECTIVES: Denture stomatitis is prevalent in older people and poses serious health risks. Ready-to-use (RTU) neutral-pH Electrolysed Oxidizing Water (EOW) is an effective environmental disinfectant used in residential care settings and geriatric wards. However, the influence of storage on stability and effectiveness for denture disinfection has not been established. This research investigated the storage-related stability and antimicrobial activity of RTU EOW, and its efficacy against Candida albicans biofilms formed on denture resin. METHODS: The pH, oxidation/reduction potential (mV), available chlorine content (mg/L) and [HOCl] (mM) of RTU EOW (Envirolyte, New Zealand) solutions (n = 22) were measured from bottle opening to 28 days following storage at 4 °C, room temperature (RT) or 37 °C. Staphylococcus aureus and C. albicans cells were incubated in 80% EOW for contact times (CTs) up to 15 min and colony-forming units (cfu) determined. Minimum inhibitory concentrations (MIC90 EOW-HOCl) after CTs up to five minutes were determined for S. aureus and C. albicans reference strains and clinical isolates. C. albicans-denture resin disc biofilms were assessed after a five-minute CT with undiluted EOW by XTT-metabolic activity assay. RESULTS: [HOCl] remained stable when RTU EOW was stored at 4 °C or RT for five months after manufacture. One-minute CT resulted in log10 cfu reductions of >6 for S. aureus and >5 for C. albicans. Mean MIC90 for five-minute CT was 37 µM (S. aureus) and 54 µM (C. albicans). Undiluted EOW reduced C. albicans biofilm metabolic activity by 86%. CONCLUSIONS: RTU neutral-pH EOW is stable over five-months storage and is an effective denture disinfectant. CLINICAL SIGNIFICANCE: The efficacy of the RTU neutral EOW against C. albicans isolates and biofilms formed on denture resin surfaces supports its use as a denture disinfectant and can inform future research to assess its potential for preventing denture-related oral Candida infections in the older population, especially in resource-limited communities.

Halitosis: prevalence, risk factors, sources, measurement and treatment - a review of the literature.

Halitosis, an offensive breath odour, has multiple sources and negative impacts on people's social interactions and quality of life. It is important for health care professionals, including general physicians and dental professionals, to understand its aetiology and risk factors in order to diagnose and treat patients appropriately. In this study, we have reviewed the current literature on halitosis regarding its prevalence, classification, risk factors, sources, measurement and treatment.

Genetic Polymorphisms in FGFR2 Underlie Skeletal Malocclusion.

Fibroblast growth factor receptor 2 (FGFR2) in craniofacial bones mediates osteoprogenitor proliferation, differentiation, and apoptosis. The distortion of proper craniofacial bone growth may cause class II and class III skeletal malocclusion and result in compromised function and aesthetics. Here, we investigated the association between variations in FGFR2 and skeletal malocclusions. First, 895 subjects were included in a 2-stage case-control study with independent populations (stage 1: n = 138 class I, 111 class II, and 81 class III; stage 2: n = 279 class I, 187 class II, and 99 class III). Eight candidate single-nucleotide polymorphisms (SNPs) in FGFR2 were screened and validated. Five SNPs (rs2162540, rs2981578, rs1078806, rs11200014, and rs10736303) were found to be associated with skeletal malocclusions (all P < 0.05). That is, rs2162540 was significantly associated with skeletal class II malocclusion, while others were associated with skeletal class III malocclusion. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis showed that the common genotypes of rs2981578 and rs10736303 contained the binding sites of RUNX2 and SMAD4. Compared with the common genotypes, the minor genotypes at these 2 SNPs decreased the binding affinity and enhancer effect of RUNX2 and SMAD4, as well the levels of FGFR2 expression. In addition, FGFR2 expression contributed positively to osteogenic differentiation in vitro. Thus, we identified FGFR2 as a skeletal malocclusion risk gene, and FGFR2 polymorphisms regulated its transcriptional expression and then osteogenic differentiation.

Simultaneous wireless assessment of intra-oral pH and temperature.

OBJECTIVES: Intra-oral pH plays an important role in the pathogenesis of tooth erosion and decay, but there is limited information about its variation in real life settings. The aims of this research were to: 1) develop a wireless device, which can be used to continuously monitor intra-oral pH and temperature in real-time; 2) test and validate the device under controlled laboratory conditions; and 3) collect data in a natural environment in a sample of healthy volunteers. METHODS: A wireless device for measuring pH and temperature simultaneously was developed, calibrated and validated against the gold standard glass electrode pH meter. A smart phone was used as data logger. The wireless device was embedded in an oral appliance and worn by eleven participants (mean age 31.1±6.9years) for 24h, while conducting standardised drinking tasks and regular daily activities. RESULTS: The wireless device could accurately measure pH and temperature both in vitro and in vivo. The recovery time following the swallow of a standard acidic drink varied markedly among individuals (mean=1.3±0.9min). The intra-oral pH and temperature recorded in the natural environment also showed a large inter- and intra-individual variability. The average intra-oral pH when asleep (6.7±0.5) was lower (p<0.001) than when awake (7.2±0.5). The average intra-oral temperature during sleep (35.6±0.5°C) was higher (p<0.001) than when awake (34.5±0.7°C). CONCLUSIONS: Intra-oral pH and temperature can be continuously and wirelessly assessed in real-life settings, and show individual-specific patterns with circadian variations. Intra-oral pH becomes slightly acidic during sleep while intra-oral temperature increases and fluctuates less. CLINICAL SIGNIFICANCE: We propose a wireless device that is capable of measuring intra-oral pH over a 24-h period. We found marked inter-individual variation after acidic stimuli, and day to sleep time variation of both intra-oral temperature and pH. Our approach may provide new insight into the relationship between oral pH, tooth wear and decay.

Secretory component mediates Candida albicans binding to epithelial cells.

OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Learning the ABC of oral fungal drug resistance.

ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals.

In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism.

Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.

Detection of Candida albicans ADH1 and ADH2 mRNAs in human archival oral biopsy samples.

OBJECTIVES: The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia. MATERIALS AND METHODS: Archival FFPE samples were obtained from four sample groups: normal oral mucosa, non-dysplastic leukoplakia, chronic hyperplastic candidosis (CHC), and non-CHC dysplastic leukoplakia. The presence of C. albicans was determined by periodic acid Schiff staining and by immunocytochemistry. C. albicans ADH1 and ADH2 mRNAs were detected using reverse transcription PCR. RESULTS: Candida albicans was detected in FFPE samples diagnosed as CHC (the histological diagnoses had been made by specialist oral pathologists, using uniform criteria), but not in any other sample group, including the non-dysplastic leukoplakias. RT-PCR confirmed a significant correlation between the expression of CaADH1 mRNA (P = 0.000), but not for CaADH2 mRNA (P = 0.056) in archival FFPE samples (n = 31) from biopsies of leukoplakia. CONCLUSIONS: Candida albicans was the predominant species in the lesions diagnosed as CHC, and the presence of C. albicans in CHC lesions was associated with a high expression of C. albicans ADH1 mRNA. There was no association between the presence of Candida and malignant transformation in the cases examined; however, the number of cases was limited and further studies are needed to further elucidate the role of C. albicans ADH1 in the pathogenesis of oral squamous cell carcinoma.

Adherence of Candida albicans to silicone is promoted by the human salivary protein SPLUNC2/PSP/BPIFA2.

Interactions between Candida albicans, saliva and saliva-coated oral surfaces are initial events in the colonization of the oral cavity by this commensal yeast, which can cause oral diseases such as candidiasis and denture stomatitis. Candida albicans also colonizes silicone voice prostheses, and the microbial biofilm formed can impair valve function, necessitating frequent prosthesis replacement. We have previously shown that saliva promoted binding of C. albicans cells to silicone in vitro, and that the selective binding of specific salivary proteins to voice prosthesis silicone mediated attachment of C. albicans cells. The C. albicans cells adhered to a polypeptide (or polypeptides) of ~36 kDa eluted from saliva-treated silicone. We show here that a protein of similar size was identified in replicate blots of the eluate from saliva-treated silicone when the blots were probed with antibodies to human SPLUNC2, a salivary protein with reported microbial agglutination properties. In addition, SPLUNC2 was depleted from saliva that had been incubated with silicone coupons. To determine whether SPLUNC2 is a yeast-binding protein, SPLUNC2 cDNA was expressed in Escherichia coli. Purified recombinant His-tagged protein (SPLUNC2r) bound to silicone as demonstrated by immunoblot analysis of an eluate from SPLUNC2r-treated silicone coupons and (35) S-radiolabelled C. albicans cells adhered in a dose-dependent manner to SPLUNC2r-coated silicone. We conclude that SPLUNC2 binds to silicone and acts as a receptor for C. albicans adherence to, and subsequent colonization of, voice prosthesis silicone.

Efficacy of air/water syringe tip sterilization.

BACKGROUND: Dental procedures involve contact between instruments and the patient's tissues, blood or saliva. This study evaluated the efficacy of the standardized sterilization of non-disposable air/water syringe tips and corrosion and contaminant build-up in these tips. METHODS: The bacterial contamination of single-use and multiple-use non-disposable air/water syringe tips after routine use and sterilization was compared to that of single-use disposable tips by microbial culturing on PCA and blood agar plates. The effect of flushing the syringe tips prior to sterilization was also measured. The amount of corrosion in single-use and multiple-use non-disposable syringes was measured by SEM and EDS analyses. RESULTS: Non-disposable syringe tips had significantly (p < 0.05) greater bacterial contamination than single-use disposable tips. There were no statistically different levels of contamination between flushed and non-flushed non-disposable syringes or between single-use and multiple-use non-disposable syringes. SEM and EDS analyses showed greater evidence of corrosion and contaminant build-up in multiple-use syringes compared to single-use non-disposable syringes. CONCLUSIONS: Sterilization of non-disposable air/water syringes is not completely effective and rinsing, or the number of uses, does not affect the effectiveness of sterilization. There may be a lower risk of cross-infection from the use of disposable air/water syringe tips, instead of non-disposable ones.

Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity.

OBJECTIVES: To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans. METHODS: The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity. RESULTS: DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values. CONCLUSIONS: A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.

Overexpression of Candida albicans CDR1, CDR2, or MDR1 does not produce significant changes in echinocandin susceptibility.

The micafungin and caspofungin susceptibilities of Candida albicans laboratory and clinical isolates and of Saccharomyces cerevisiae strains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicans gave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2 showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1 or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1 showed slightly decreased susceptibility to micafungin in these assays. A C. albicans clinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.

Characterization of two Candida albicans surface mannoprotein adhesins that bind immobilized saliva components.

Salivary components, including the basic proline-rich proteins (bPRP), act as receptors for the adherence of Candida albicans, and this interaction may be important for oral colonization and the development of mucosal Candida infections. Treatment of C. albicans cells with agents affecting either proteinacious or carbohydrate surface macromolecules reduced their adherence in in vitro assays of C. albicans adherence to saliva-coated hydroxyapatite beads and to membrane-immobilized salivary bPRP. In order to identify C. albicans adhesins that bind saliva receptors, yeast cell surface material was extracted by mild glucanase treatment, and was shown to competitively inhibit ( > 50%) the adherence of C. albicans yeast cells in both assays. Concanavalin A sepharose affinity chromatography was used to partially purify glycosylated components of the extract, and two polypeptides (97.4 and 35 kDa) were further purified by preparative SDS PAGE separation and electro-elution. The 97.4 and 35 kDa polypetides each possessed greater adherence-inhibitory specific activity (> 100-fold and > 30-fold respectively) than the original glucanase extract from C. albicans yeast cells. The 35 kDa putative surface protein was identified by N-terminal sequencing and immunoblotting, as the 1,3-beta glucosyltransferase, Bgl2p.

Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative.

Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5(+)) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 microg ml(-1)), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 microg ml(-1)). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer D-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted D-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC(50)], 4 microM) which chemosensitized AD/PDR5(+) to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC(50), 30 microM) and Cdr2p (IC(50), 2 microM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter Ben(R)p, or the FLC drug target lanosterol 14 alpha-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC(50), 1 microM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

Saliva promotes Candida albicans adherence to human epithelial cells.

Adhesion of Candida cells to oral surfaces is an initial event in pathogenesis. Since specific immobilized salivary components mediate the binding of Candida albicans to hydroxyapatite, we hypothesized that saliva may also promote adherence to oral epithelia via a similar mechanism. In an in vitro model, C. albicans ATCC 10261 yeast cells adhered in a saturable manner to monolayers of three cultured human epithelial cell lines (A549, HEp-2, and HET-1A). The addition of whole saliva to the assay promoted the binding of C. albicans to all cell lines in a dose-dependent manner, but pre-incubation of the epithelial cells with pooled whole saliva had no effect on subsequent adherence. Pre-incubation of the yeast cells with pooled whole saliva, however, significantly enhanced (by up to 120%, P < 0.05) binding to epithelial cell monolayers, and pooled saliva that had been pre-incubated with C. albicans yeast cells was defective in promoting yeast adherence. There was a negative correlation (r = 0.68, P < 0.005) between specific IgA titers against whole cells of C. albicans and adherence-promoting activities for individual saliva samples. The adhesion-inhibitory effect of specific anti-C. albicans IgA was reversed by depletion of IgA from saliva by affinity chromatography. Factors in whole saliva, therefore, bound to the yeast cells, counter the C. albicans-specific salivary IgA inhibitory effect on adhesion and promote the adherence of C. albicans yeast cells to cultured epithelial cells.

Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters.

Analysis of the transport functions of individual Candida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic PDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u(-)) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u(-) was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC(80)s] were 0.625 microg/ml for fluconazole, <0.016 microg/ml for ketoconazole, and <0.016 microg/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC(80)s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 microg/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicans Cdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.

Distinguishing Candida species by beta-N-acetylhexosaminidase activity.

A variety of fungi produce the hydrolytic enzyme beta-N-acetylhexosaminidase (HexNAcase), which can be readily detected in assays by using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as a substrate. In the present study we developed a microtiter plate-based HexNAcase assay for distinguishing Candida albicans and Candida dubliniensis strains from other yeast species. HexNAcase activity was detected in 89 of 92 (97%) C. albicans strains and 4 of 4 C. dubliniensis strains but not in 28 strains of eight other Candida species, 4 Saccharomyces cerevisiae strains, or 2 Cryptococcus neoformans strains. The HexNAcase activity in C. albicans and C. dubliniensis was strain specific. All except three clinical C. albicans isolates among the C. albicans strains tested produced enzyme activity within 24 h. These strains did produce enzyme activity, however, after a prolonged incubation period. For two of these atypical strains, genomic DNA at the C. albicans HEX1 gene locus, which encodes HexNAcase, showed nucleotide differences from the sequence of control strains. Among the other Candida species tested, only C. dubliniensis had a DNA sequence that hybridized with the HEX1 probe under low-stringency conditions. The microtiter plate-based assay used in the present study for the detection of HexNAcase activity is a simple, relatively inexpensive method useful for the presumptive identification of C. albicans and C. dubliniensis.